B lymphocytes are the origin of humoral immunity, represent a substantial portion of hematopoietic malignancies, and contribute to autoimmunity. Consequently, cell surface molecules expressed by B cells and their malignant counterparts are important targets for immunotherapy. CD20, a B cell-specific member of the MS4A gene family, is expressed on the surface of immature and mature B cells and their malignant counterparts (Tedder and Engel (1994) Immunol. Today 15:450-454).
A limited analysis of CD20 transcripts in mouse cell lines and tissues suggests that mouse CD20 is also B cell-specific (Tedder, et al. (1988) J. Immunol. 141:4388). Both human and mouse CD20 cDNAs encode a membrane-embedded protein with hydrophobic regions of sufficient length to pass through the membrane four times (Tedder, et al. (1988) J. Immunol. 141:4388; Tedder, et al. (1988) Proc. Natl. Acad. Sci. USA. 85:208; Einfeld, et al. (1988) EMBO J. 7:711; Stamenkovic and Seed (1988) J. Exp. Med. 167:1975). Mouse and human CD20 are well conserved (73%) in amino acid sequence, particularly the transmembrane and long amino- and carboxyl-terminal cytoplasmic domains (Tedder, et al. (1988) J. Immunol. 141:4388). The cytoplasmic domains are serine- and threonine-rich with multiple consensus sequences for phosphorylation. Human CD20 is not glycosylated, but three isoforms (33-, 35- and 37,000 Mr) result from the differential phosphorylation of a single protein on different serine and threonine residues (Tedder, et al. (1988) Molec. Immunol. 25:1321; Tedder and Schlossman (1988) J. Biol. Chem. 263:10009; Valentine, et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84:8085).
CD20 plays a role in the regulation of human B cell activation, proliferation and Ca2+ transport (Tedder, et al. (1985) J. Immunol. 135:973; Bubien, et al. (1993) J. Cell Biol. 121:1121). Antibody ligation of CD20 can generate transmembrane signals that result in enhanced CD20 phosphorylation (Tedder and Schlossman (1988) J. Biol. Chem. 263:10009), induction of c-myc and B-myb oncogene expression (Smeland, et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:6255; Golay, et al. (1992) J. Immunol. 149:300), induced serine/threonine and tyrosine phosphorylation of cellular proteins (Deans, et al. (1993) J. Immunol. 151:4494), increased CD18, CD58 and MHC class II molecule expression (White, et al. (1991) J. Immunol. 146:846; Clark and Shu (1987) J. Immunol. 138:720), and protein tyrosine kinase activation that induces B cell adhesion (Kansas and Tedder (1991) J. Immunol. 147:4094). CD20 ligation promotes transmembrane Ca2+ transport (Bubien, et al. (1993) J. Cell Biol. 121:1121), but does not usually lead to increased intracellular calcium ([Ca2+]i)3 levels (Bubien, et al. (1993) J. Cell Biol. 121:1121; Tedder, et al. (1986) Eur. J. Immunol. 16:881; Golay, et al. (1985) J. Immunol. 135:3795), except after extensive crosslinking (Deans, et al. (1993) J. Immunol. 151:4494). Antibody binding to CD20 inhibits B cell progression from the G1 phase into the S/G2+M stages of cell cycle following mitogen stimulation, and inhibits mitogen-induced B cell differentiation and antibody secretion (Tedder, et al. (1985) J. Immunol. 135:973; Tedder, et al. (1986) Eur. J. Immunol. 16; Golay, et al. (1985) J. Immunol. 135:3795; Golay and Crawford (1987) Immunology 62:279). Extensive CD20 cross-linking can also influence apoptosis (Holder, et al. (1995) Eur. J. Immunol. 25:3160; Shan, et al. (1998) Blood 91:1644). These divergent observations may be explained in part by the finding that CD20 is a component of an oligomeric complex that forms a membrane transporter or Ca2+ ion channel that is activated during cell cycle progression (Bubien, et al. (1993) J. Cell Biol. 121:1121; Kanzaki, et al. (1995) J. Biol. Chem. 270:13099; Kanzaki, et al. (1997) J. Biol. Chem. 272:14733; Kanzaki, et al. (1997) J. Biol. Chem. 272:4964). Despite this, B cell development and function in a line of CD20-deficient (CD20−/−) mice is reported to be normal (O'Keefe, et al. (1998) Immunogenetics 48:125).
The majority of human B cell-lineage malignancies express CD20 (Anderson, et al. (1984) Blood 63:1424). Chimeric or radiolabeled monoclonal antibody-based therapies directed against CD20 have been used for non-Hodgkin's lymphoma (Press, et al. (2001) Hematology: 221-240; Kaminski, et al. (1993) N. Engl. J. Med. 329:459-465; Weiner (1999) Semin. Oncol. 26:43-51; Onrust, et al. (1999) Drugs 58:79-88; McLaughlin, et al. (1998) Oncology 12:1763-1769). Clinical studies indicate that anti-CD20 monoclonal antibody therapy also ameliorates the manifestations of rheumatoid arthritis, idiopathic thrombocytopenic purpura and hemolytic anemia, as well as other immune-mediated diseases (Silverman and Weisman (2002) Arthritis Rheum. 48:1484-1492; Edwards and Cambridge (2001) Rheumatology 40:1-7).
Competing hypotheses are employed to explain the therapeutic efficacy of anti-CD20 monoclonal antibodies in vivo. In one model, CD20 serves as a membrane-embedded target for monoclonal antibody-mediated depletion of B cells through activation of the innate immune system or the initiation of effector mechanisms (Reff, et al. (1994) Blood 83:435-445; Maloney, et al. (1997) Blood 90:2188-2195; Maloney, et al. (1997) J. Clin. Oncol. 15:3266-3274).
Rituximab, a chimeric human IgG1 anti-human CD20 monoclonal antibody is highly effective in inducing classical pathway complement (C) activation and C-dependent cytotoxicity of freshly isolated lymphoma cells and B cell lines (Reff, et al. (1994) Blood 83:435-445; Golay, et al. (2001) Blood 98:3383-3389; Cragg, et al. (2003) Blood 101:1045-1052; Di Gaetano, et al. (2003) J. Immunol. 171:1581-1587; Bellosillo, et al. (2001) Blood 98:2771-2777). Rituximab also activates C in vivo in both patients (van der Kolk, et al. (2001) Br. J. Hematol. 1115:807-811) and primates (Kennedy, et al. (2003) Blood 101:1071-1079). Furthermore, tumor cell expression of C regulatory proteins, including CD59, is associated with resistance to anti-CD20 therapy (Golay, et al. (2001) Blood 98:3383-3389; Treon, et al. (2001) J. Immunotherapy 24:263-271). Although many consider C-dependent cytotoxicity to be the major pathway used by Rituximab antibody to deplete human lymphoma cells in vitro and in vivo (Golay, et al. (2001) Blood 98:3383-3389; Cragg, et al. (2003) Blood 101:1045-1052; Di Gaetano, et al. (2003) J. Immunol. 171:1581-1587; Golay, et al. (2000) Blood 95:39003908; Di Gaetano, et al. (2001) Br. J. Hematol. 114:800-809; Weiner (2003) Blood 101:788), others have found that the susceptibility of tumor cells to C-mediated lysis and expression of C inhibitors CD46, CD55, and CD59 on tumor cells does not predict the outcome of Rituximab therapy (Weng and Levy (2001) Blood 98:1352-1357). Other antibody-dependent effects also appear important since a chimeric anti-CD20 monoclonal antibody of an isotype different than that used clinically does not deplete normal B cells in non-human primates (Anderson, et al. (1997) Biochem. Soc. Transac. 25:705-708) and the anti-tumor effect of anti-CD20 monoclonal antibody depends in part on immune activation through Fc receptors (FcγR) for IgG (Clynes, et al. (2000) Nature Med. 6:443-446). Alternatively, anti-CD20 monoclonal antibody treatment alters transmembrane Ca2+ transport and B cell function, which disrupts progression through cell cycle (Tedder and Engel (1994) Immunol. Today 15:450-454) and can induce B cell apoptosis (Shan, et al. (1998) Blood 91:1644-1652; Demidem, et al. (1997) Cancer Biother. Radiopharm. 12:177-186).
It is difficult to differentiate between these hypotheses in vivo due to the complexities of carrying out mechanistic studies in humans undergoing immunotherapy (Edwards and Cambridge (2001) Rheumatology 40:1-7). Moreover, human studies primarily focus on changes in blood, which contains <2% of the B cells outside of the bone marrow. Thus, it is difficult to accurately ascertain the effects of anti-CD20 therapies on the majority of B cells, which are found in peripheral lymphoid tissues.
Needed in the art are improved reagents and methods for altering B cell function, in particular in B cell disorders such as B cell malignancies and autoimmune diseases. Also needed are new anti-CD20 monoclonal antibodies with different immunoreactive characteristics than conventional monoclonal antibodies directed against CD20.